Janus kinase (JAK) is a family of intracellular non-receptor tyrosine kinases, ranging from 120-140 kDa, that transduce cytokine-mediated signals via the JAK-STAT pathway. The JAK family plays a role in the cytokine-dependent regulation of proliferation and function of cells involved in immune response. Currently, there are four known mammalian JAK family members: JAK1, JAK2, JAK3 and TYK2.
JAK1, JAK2 and TYK2 are ubiquitously expressed whereas JAK3 is expressed in the myeloid and lymphoid lineages. The JAK family members are non-receptor tyrosine kinases that associate with many hematopoietin cytokines, receptor tyrosine kinases and GPCR's. JAK1−/− mice were found to be developmentally similar to the JAK1+/+ although they weighed 40% less than the wild-type and failed to nurse at birth. These pups were not viable and died within 24 hours of birth (Meraz et al Cell, 1998, 373-383). JAK1 deficiency led to reduced number of thymocytes, pre-B cells and mature T and B lymphocytes. TYK2(−/−) mice, on the other hand, are viable, demonstrating subtle defects in their response to IFN-α/β and IL-10 and profound defects to the response of IL-12 and LPS.
The breast cancer susceptibility protein (BRCA1) acts as a tumor suppressor and contributes to cell proliferation, cycle regulation, as well as DNA damage and repair. BRCA1 (−/−) mice develop normally but die by 7.5 days post embryo suggesting a key role of BRCA1 for development. Mice in which the BRCA1 protein was overexpressed led to inhibition of cell growth and sensitized cells to cytotoxic reagents. In the human prostate cancer cell line Du-145 (Gao FEBS Letters 2001, 488, 179-184), enhanced expression of BRCA1 was found to correlate with constitutive activation of STAT3 as well as activation of JAK1 and JAK2. Moreover, antisense oligonucleotides selective for STAT3 led to significant inhibition of cell proliferation and apoptosis in Du-145 cells. This data supports the potential utility of JAK1 and JAK2 inhibitors in the treatment of prostate cancer.
Campbell et al (Journal of Biological Chemistry 1997, 272, 2591-2594) as reported that STAT3 is constitutively activated v-Src transformed cells. To test whether STAT3 activation resulted via signaling through the JAK-STAT pathway, three fibroblast cell lines (NIH3T3, Balb/c, and 3Y1) were transformed with v-Src. The level of JAK1 phosphorylation in NIH3T3 cells was markedly increased in cells overexpressed with v-Src or mutant c-Src (Y527F) compared to those in the less transforming c-Src. This result correlated with increased JAK1 enzymatic activity. Similar results were observed with JAK2 albeit to a lesser extent. These results are consistent with constitutive activation of JAK1 and possibly JAK2 which contribute to the hyperactivation of STAT3 in Src-transformed cells.
Asthma is a disease that is increasing in prevalence and results in “airway obstruction, airway hyperresponsiveness, and airway inflammation and remodeling” (Pernis The Journal of Clinical Investigation 2002, 109, 1279-1283). A common cause is the inappropriate immune responses to environmental antigens usually involving CD4+ T helper cells (TH2) which are triggered from cytokines IL-4, IL-5, IL-6, IL-10, and IL-13 which signal through JAK1/JAK3-STAT6 pathway. Th1 cells are thought to be involved with the “delayed-type hypersensitivity responses” which secrete IL-2, IFN-γ, and TNF-β and signal through the JAK2/TYK2-STAT4 pathway. STAT6 (−/−) mice were protected from AHR when challenged with environmental antigens and showed no increase in IgE levels or the quantity of mucous containing cells.
JAK2 is a cytoplasmic protein-tyrosine kinase that catalyzes the transfer of the gamma-phosphate group of adenosine triphosphate to the hydroxyl groups of specific tyrosine residues in signal transduction molecules. JAK2 mediates signaling downstream of cytokine receptors after ligand-induced autophosphorylation of both receptor and enzyme. The main downstream effectors of JAK2 are a family of transcription factors known as signal transducers and activators of transcription (STAT) proteins. Studies have disclosed an association between an activating JAK2 mutation (JAK2V617F) and myeloproliferative disorders. The myeloproliferative disorders, a subgroup of myeloid malignancies, are clonal stem cell diseases characterized by an expansion of morphologically mature granulocyte, erythroid, megakaryocyte, or monocyte lineage cells. Myeloproliferative disorders (MPD) include polycythemia vera (PV), essential thrombocythemia (ET), myeloid metaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), juvenile myelomonocytic leukemia (JMML) and systemic mast cell disease (SMCD). It has been suggested that abnormalities in signal transduction mechanisms, including constitutive activation of protein tyrosine kinases, initiate MPD.
JAK3 associates with the common gamma chain of the extracellular receptors for the following interleukins: IL-2, IL-4, IL-7, IL-9 and IL-15. A JAK3 deficiency is associated with an immune compromised (SCID) phenotype in both rodents and humans. The SCID phenotype of JAK3−/− mammals and the lymphoid cell specific expression of JAK3 are two favorable attributes of a target for an immune suppressant. Data suggests that inhibitors of JAK3 could impede T-cell activation and prevent rejection of grafts following transplant surgery, or to provide therapeutic benefit to patients suffering autoimmune disorders.
3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a 556-amino acid containing enzyme comprised of a C-terminal Pleckstrin homology (PH) domain (residues 459-550) and an N-terminal kinase domain (residues 70-359). The PH domain of PDK1 binds phosphatidylinositols (e.g., phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) produced by phosphatidylinositol kinases, such as phosphatidylinositol 3-kinase (PI3K) and whose levels are, in part, controlled by phosphatases such as PTEN (phosphatase and tensin homologue). PDK1 plays a central role in the PI3K/Akt pathway and has been called a “master regulator” kinase due to its role as a critical upstream activating kinase that phosphorylates the so-called T-loop phosphorylation site for multiple kinases in the AGC family of kinases including but not limited to all three isoforms of PKB (PKBα, PKBβ, PKBγ, also known as Akt1, Akt2, and Akt3, respectively), RSK (three isoforms RSK1, RSK2, RSK3, also known as p90RSK), p70S6K (two isoforms, S6K1 and S61 (2), PKN (three isoforms PKN1, PKN2, and PKN3), SGK1 and PKC.
Signals from several peptide growth factors including insulin, insulin-like growth factor-1, vascular endothelial growth factor and platelet-derived growth factor are transduced by PKB. Like PDK1, PKB contains a PH domain that binds phosphatidyl 3,4,5-triphosphate. PKB is translocated to the plasma membrane and phosphorylated by PDK1 at residue T-308/309 (the two phosphosites correspond to different human isoforms) in response to the second messenger phosphatidyl 3,4,5-triphosphate produced by PI3K. Activation of PKB in tumor cells results in increased cellular survival via anti-apoptotic signals and also proliferation. PKBβ amplification has been observed in a proportion of several tumor types including ovarian, breast and pancreatic cancers. Similarly, PKBα amplification has been observed in a percentage of gastric adenocarcinoma samples. Recently, an activated mutant form of PKBα (E17K) was detected in a number of breast (8%), colorectal (6%), and ovarian (2%) cancers. PDK1 kinase inhibitors are useful as treatments for diseases linked to PKB signaling (such as cancer, Cowden syndrome, Lhermitte-Dudos disease and Bannayan-Zonana syndrome) by preventing activation of PKB signaling by PDK1.
Similarly, PDK1 kinase inhibitors are useful for treating cancer or other proliferative disorders by blocking the activation of p70S6K by PDK1. There are several substrates of p70S6K, such as ribosomal S6 protein, eIF4B, PDCD4 etc., that are involved in translation inhibition complex formation or ribosomal protein synthesis Inhibition of protein synthesis via inhibition of phosphorylation of ribosomal S6 protein is believed to inhibit the proliferation of tumor cells by mTOR inhibitors (e.g., rapamycin). p70S6K gene amplification has been observed in breast tumor specimens, simultaneous amplification of p70S6K and HER-2 correlates with poor survival in cancer patients. Hyperactivation of p70S6K (as measured by phosphorylation of T389) has been observed by immunohistochemical analysis of breast, head and neck squamous cell carcinoma (HNSCC), glioblastoma, lung and liver primary tumor specimens.
Likewise, PDK1 kinase inhibitors are useful for the treatment of cancer by blocking the activation of RSK1 (also known as p90RSK) by PDK1. RSK1 transduces anti-apoptotic and proliferative signals be mediating phosphorylation directly or indirectly of BAD, LKB1, TSC2, NFkB, mTOR. Ras/MAPK pathway is activated in >50% of primary tumors. RSK1 activity is correlated with MAPK activity. RSK1 is overexpressed in primary breast and prostate cancer samples.
PDK1 signaling regulates multiple critical steps in angiogenesis. Inhibitors of the activity of PDK1 are thus useful in the treatment of cancer (both primary tumors and metasteses).
In particular, PDK1 is a key signaling molecule in cancers associated with deregulated activity of the PTEN/PI3K pathway including, but not limited to PTEN loss of function mutations, PI3K gain of function mutations and receptor tyrosine kinase gain of function mutations. Thus, inhibitors of PDK1 are especially useful in the treatment of cancers with aberrant signaling of the PTEN/PI3K pathway.
PDK1 signaling has also been implicated in tumorigenesis and a PDK1 inhibitor is useful for the tumor prevention or for the prevention of tumor recurrence. Mice with a PTEN heterozygous (PTEN+/−) genotype are well-known to spontaneously develop tumors. Alessi and co-workers found that PDK1 hypomorphic PTEN+/− mice expressing <30% of normal PDK1 protein levels showed a significant delay in tumor formation as compared to littermate controls expressing normal levels of PDK1 protein (Current Biology, 2005, 15, 1839-1846)
SGK1 (serum and glucocorticoid-regulated kinase-1) activity is critical for insulin-mediated Na+ retention and hypertensive effects Inhibition of SGK1 activity by a PDK1 kinase inhibitor is useful in treating hypertension and/or hypoinsulinemia.